FIG 5.

Identification of mutations in NS4A conferring resistance to SBI-0090799 and inhibition of ZIKV replication organelle formation by this compound. (A) Huh-7.5 cells were pretreated for 16 h with increasing concentrations of SBI-0090799 or DMSO and then infected with ZIKV MR766 selected for 5 passages in the presence of either DMSO (black line) or SBI-0090799 (10 μM) (cyan line). Twenty-four hours after infection, cells were fixed and analyzed by immunofluorescence imaging. For each condition, the percentage of infection was calculated as the ratio of the number of infected cells stained for ZIKV envelope protein to the number of cells stained with DAPI. Data represent the means ± SEM from n = 3, normalized to mean values for DMSO-treated wells. Curves were fitted by nonlinear regression analysis. (B) Sequence alignment for NS4A N-terminal region. Red squares indicate the amino acid residues replaced after 5 passages of viral selection in the presence of SBI-0090799. Arrows indicate the replacing residues. (C) Huh-7.5 cells were electroporated with RNA encoding the replicon pZIKV Rep, bearing the indicated mutation in NS4A, or the corresponding sequence revertant to wild type (rev). The antiviral activity of SBI-0090799 was assessed 24 h postelectroporation by measuring the Renilla luciferase activity as a readout of viral replication. Renilla counts at 24 h were normalized to the corresponding values at 4 h posttransfection, reflecting electroporation efficiency. Data represent the means ± SEM from n = 3, with each replicon normalized to its mean values for DMSO-treated wells. Curves were fitted by nonlinear regression analysis. (D) Huh-7.5 cells were infected for 1 h at 37°C with ZIKV MR766 at an MOI of 1. After removal of the inoculum and two washes with PBS, fresh medium supplemented with either DMSO or SBI-0090799 (10 μM) was added to each well. At the indicated time postinfection, cells were harvested and a Western blot was performed. Replication was quantified by immunoblotting of NS3 and NS5, and relative quantification for these two proteins is indicated. Asterisk indicates a nonspecific band recognized by the NS5A antiserum. An additional nonspecific band recognized by the NS5 antiserum is shown as a loading control. (E) Huh-7.5 cells were infected for 1 h at 37°C with ZIKV MR766 at an MOI of 1. After removal of the inoculum, fresh medium supplemented with either DMSO, SBI-0090799 (10 μM), or the RNA translation inhibitors cycloheximide (50 μg/ml) or emetine (2 μM) was added to each well. At the indicated time postinfection, cells were harvested, and intracellular RNA was isolated and quantified by RT-qPCR. For each condition, ZIKV RNA levels relative to the DMSO-treated cells are shown for n = 3 biological replicates. RPLP0 was used as a housekeeping gene. (F and G) SBI-0090799 inhibits ZIKV replication organelle formation. (F) Transmission electron microscopy images of 70-nm sections of resin-embedded Huh7/Lunet-T7 cells transfected for 18 h with pIRO-Z in the presence of either SBI-0090799 (12.5 μM) or DMSO (0.125%, vol/vol). Lower panels are magnifications of yellow-squared areas in the upper panels. Scale bars, 500 nm and 200 nm in the upper and lower panels, respectively. Yellow arrows indicate VPs or ER membrane in the case of mock-infected cells. (G) For determining the number of pIRO-Z-induced vesicle packets (VPs) per cell, 20 cell profiles were randomly selected, and VPs were manually counted. Means ± SEM are shown for n = 3 independent experiments. Differences in numbers of vesicle packets per cell were analyzed using the two-tailed unpaired t test with Welch’s correction. ****, P < 0.0001.